BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase B M B 400, Part Three Gene Expression and Protein Synthesis Lecture Notes Overview of Part Three: The pathway of gene expression Recall the Central Dogma of molecular biology: DNA is transcribed into RNA, which is translated into protein. We will cover the material in that order, since that is the direction that information flows. However, there are additional steps, in particular the primary transcript is frequently a precursor molecule that is processed into a mature RNA. E.g., the rRNA genes are transcribed into pre-rRNA, that is cleaved, methylated and modified to produce mature rRNA. Many mRNAs, especially in eukarytoes, are derived from pre-mRNAs by splicing and other processing events. This general topic will be covered after transcription and before translation. Fig. 3.1.1 BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase CHAPTER 10= PART THREE-I. TRANSCRIPTION: RNA polymerase A. RNA polymerase catalyzes the DNA-dependent synthesis of RNA 1. RNA polymerase requires DNA as a template. In duplex DNA, the template strand of DNA is copied into RNA by RNA polymerase. The choice of nucleotides during this process is directed by base complementarity, so that the sequence of RNA synthesized is the reverse complement of the DNA template strand. It is the same sequence as the nontemplate (or top) strand, except that U's are present instead of T's. This process of RNA synthesis directed by a DNA template, catalyzed by RNA polymerase, is called transcription. 2. RNA polymerase does not require a primer to initiate transcription. 3. RNA polymerase catalyzes the sequential addition of a ribonucleotide to the 3' end of a growing RNA chain, with the sequence of nucleotides specified by the template. The substrate NTP is added as a NMP with the liberation of pyrophosphate. This process occurs cyclically during the elongation phase of transcription. NTP + (NMP)n ←→ (NMP) n+1 + PPi template DNA Mg++ Fig. 3.1.2. Sequential addtion of ribonucleotides to growing RNA BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. The liberated pyrophosphate is cleaved in the cell to 2 Pi, an energetically favorable reaction that drives the reaction in the direction of synthesis. 5. In the presence of excess PPi, the reverse reaction of pyrophosphorolysis can occur. 6. Synthesis always proceeds in a 5' to 3' direction (with respect to the growing RNA chain). The template is read in a 3' to 5' direction. B. E. coli RNA polymerase structure 1. This one RNA polymerase synthesizes all classes of RNA mRNA, rRNA, tRNA 2. It is composed of four subunits. a. Core and holoenzyme α2ββ'σ ←→ α2ββ' + σ Holoenzyme = α2ββ'σ = core + σ = can initiate transcription accurately as the proper site, as determined by the promoter Core = α2ββ' = can elongate a growing RNA chain A promoter can be defined in two ways. (a) The sequence of DNA required for accurate, specific intiation of transcription (b) The sequence of DNA to which RNA polymerase binds to accurately initiate transcription. b. Subunits Subunit Size Gene Function β' 160 kDa rpoC β' + β form the catalytic center. β 155 kDa rpoB β' + β form the catalytic center. α 40 kDa rpoA enzyme assembly; also binds UP sequence in the promoter σ 70 kDa (general) rpoD confers specificity for promoter; binds to -10 and -35 sites in the promoter Bacteria have several σ factors, ranging in size from 32 to 92 kDa, each of which confers specificity for a different type of promoter. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Fig. 3.1.3. Diagram of E. coli RNA polymerase α E. coli RNA polymerase α β σ β' UP -35 -10 3. Three-dimensional structure of E. coli RNA polymerase Crystals suitable for X-ray diffraction studies have not been obtained yet, but the surface topography can be determined from by electron crystallography of two-dimensional crystalline arrays. Fig. 3.1.4. Low resolution structure of RNA polymerases from electron crystallography e- Electron microscope Computer workstation Form a 2-dimensional crystal Record micrographs from the Average the information from (i.e. 1 molecule thick) on a layer of crystalline arrays at three angles the micrographs to determine a positively charged lipid. Place on (2 tilted to the incident electron low resolution map (e.g. 27 an electron microscope grid and beam and 1 untilted). Angtroms) of the surface stain with uranyl acetate. topography (i.e. the part outlined by the uranyl acetate. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Fig. 3.1.5. E. coli RNA polymerase core (left) and holoenzyme (right) Images from analysis by Seth Darst. The structure in the presence of σ (holoenzyme) is on the right; note the open channel for DNA binding. The structure in the absence of σ (the core enzyme) is on the left. Note that the channel is now closed, as if the fingers and thumbs of a hand now closed to make a circle. This striking conformational change that occurs when σ dissociates is thought to confer high processivity on the RNA polymerase. Fig. 3.1.6. Diagram of features of E. coli RNA polymerase holoenzyme o Holoenzyme for RNA polymerase from E. coli α 2 ββ'σ 27 A resolution View perpendicular to channel: "thumb" Similar to DNA polymerase I Klenow o 100 A Channel with probable DNA-binding and active sites. 25 Angstroms in diameter and 55 Angstroms long (enough for about 16 bp of DNA). "fingers" Darst, S.A., E.W. Kubalek & R.D. Kornberg (1989) Nature 340: 730-732. (view flipped 180 o ) o o 160 A 100 A thick BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. Assembly of E. coli RNA polymerase The α subunit has two distinct domains. The N-terminal domain (α-NTD) is involved in dimerization to form α2 and further assembly of the RNA polymerase. The C- terminal domain has different functions, being used in the binding to the UP DNA sequence at promoters for rRNA and tRNA genes and in communication with many, but not all, transcriptional activators. Fig. 3.1.7. Role of the α subunit in assembly and other functions C. E. coli RNA polymerase mechanism 1. Mode of action of σ factors The presence of the σ factor causes the RNA polymerase holoenzyme to be selective in choosing the site of initiation. This is accomplished primarily through effects on the dissociation rate of RNA polymerase from DNA. a. Core has strong affinity for general DNA sequences. The t1/2 for dissociation of the complex of core-DNA is about 60 min. This is useful during the elongation phase, but not during initiation. b. Holoenzyme has a reduced affinity for general DNA; it is decreased about 104 fold. The t1/2 for dissociation of holoenzyme from general DNA is reduced to about 1 sec. c. Holoenzyme has a greatly increased affinity for promoter sequences. The t1/2 for dissociation of holoenzyme from promoter sequences is of the order of hours. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 2. Events at initiation of transcription a. RNA polymerase holoenzyme binds to the promoter to form a closed complex; at this stage there is no unwinding of DNA. b. The polymerase-promoter complex undergoes the closed to open transition, which is a melting or unwinding of about 12 bp. c. The initiating nucleotides can bind to the enzyme, as directed by their complementary nucleotides in the DNA template strand, and the enzyme will catalyze formation of a phosphodiester bond between them. This polymerase-DNA-RNA complex is referred to as the ternary complex. d. During abortive initiation, the polymerase catalyzes synthesis of short transcripts about 6 or so nucleotides long and then releases them. e. This phase ends when the nascent RNA of ~6 nucleotides binds to a second RNA binding site on the enzyme; this second site is distinct from the catalytic center. This binding is associated with "resetting" the catalytic center so that the enzyme will now catalyze the synthesis of oligonucleotides 7-12 long. f. The enzyme now translocates to an new position on the template. During this process sigma leaves the complex. A conformational change in the enzyme associated with sigma leaving the complex lets the "thumb" wrap around the DNA template, locking in processivity. Thus the core enzyme catalyzes RNA synthesis during elongation, which continues until "signals" are encountered which indicate termination. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase Figure 3.1.8. Events at initiation BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 3. Transcription cycle a. Initiation RNA polymerase holoenzyme binds at the promoter, unwinds DNA (open complex) and form phosphodiester links between the initiating nucleotides. b. Elongation σ dissociates and core elongates. Perhaps other factors bind to enhance the processivity (maybe NusA?) c. Termination At a termination signal, RNA polymerase dissociates from the DNA template and the newly synthesized RNA is released. The factor ρ is required at many terminators. Figure 3.1.9. BMB 400 Part Three - I = Chapter 10. Transcription: RNA polymerase 4. Sites on RNA Polymerase core a. The enzyme covers about 60 bp of DNA, with a transcription bubble of about 17 bp unwound. b. The duplex DNA being transcribed is unwound at one active site on the enzyme, thereby separating the two strands (Fig. 3.1.10). The two strands are rewound at another active site, regenerating duplex DNA. c. Within the unwound region (bubble), the 3' terminus of the growing RNA chain is bound to its complement on the template strand via H-bonding.